1. Field of the Invention
The present invention relates generally to mass spectrometry. More particularly, the present invention is directed to a mass spectrometer configured to handle volatile/non volatile samples, gas and solid phase sample introduction, and ionization methods appropriate to the full spectrum of molecular masses.
2. Description of the Related Art
Rapid and accurate identification of biological agents is essential in diagnosing diseases, anticipating epidemic outbreaks, monitoring food supplies for contamination, regulating bioprocessing operations. It is highly desirable not only to rapidly distinguish between related biological agents especially pathogenic agents, but also to unmistakably identify species and strains in complex matrices in general and, particularly, for the purpose of risk assessment in field situations.
Furthermore, the real threat from biological weapons as tools of modern warfare and urban terrorism is rising. Development of early detection, counter measures, and remediation technology is a high priority in many military, government and private laboratories around the world. Biological warfare (BW) agents such as Bacillus anthracis (anthrax), Clostridium tetani (tetanus), and Clostridium botulinum (botulism) are of critical concern since these spores are non-growing, heat-resistant, dehydrated, and resistant to extremes of temperature, pH, desiccation, radiation, and chemical agents. Due to their high stability, spores are difficult to stain using typical cell biology methods and, consequently, are challenging to detect and enumerate. This stability and difficulty with conventional detection methods, in turn, make them an attractive tool for use in BW weapons.
Relatively recently, mass spectrometric techniques have been developed for generating specific protein profiles for various biological agents. Mass spectrometry is an analytical technique in which atoms or molecules from a sample are ionized (usually positively) and separated according to their mass-to-charge ratio (m/z). The resulting mass spectrum is a record of the intensity of the signal as a function of m/z. The instrument used to record a mass spectrum is called a mass spectrometer. Because every compound has a distinct (though not necessarily unique) molecular weight and fragmentation pattern, mass spectrometers have a unique potential for the broadband detection and identification of chemical and/or biological agents.
A typical spectrometer has, among others, the following essential parts: the ionizer, detector and mass analyzer frequently provided with data-handling electronics. There are a number of different techniques and solutions for each of these parts.
One of the early-developed ionization techniques was Electron Impact Ionization (EI). The principle of the EI source is shown in FIG. 1 and includes a filament 10, which serves as a source of electrons 12. A target or anode 14 is positively charged with respect to the filament 10 and attracts electrons out of it. A repeller 16 is a positively charged electrode which pushes positive ions away from the filament 10 through a lens stack 18 including a series of increasingly more negative electrodes which accelerate the positive ions in such a way that they become focused into a relatively narrow beam. When a sample molecule enters the EI source, it is hit by the electrons 12 and is ionized.
Most low molecular weight organic molecules are introduced as neutral volatile samples (generally upon heating) and are charged or ionized by the electron impact (EI) method. EI mass spectra are generally interpretable and can be used to deduce the chemical structure.
EI, however, may be limited in its use. As molecules become larger and carry more polar functional groups, they also become less volatile. However, volatility is required for EI, because it is a gas phase ionization technique. Accordingly, while EI is suitable for detection of low molecular weight chemical agents, it may not be sufficiently efficient for the larger toxins and microorganisms that comprise potential biological threat agents.
While a number of other ionization techniques have been developed over the last two decades, two new methods: electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are now the most commonly used for non-volatile biological samples.
ESI is an ionization technique for small amounts of large and/or labile molecules such as peptides, proteins, organometallics, and polymers and forms ions directly from a sprayed solution. A solution of the sample is sprayed though a needle having a certain potential, which causes the spray to be charged as it is nebulized. The droplets evaporate in a region maintained at a vacuum. As any other ionization technique, ESI may have certain limitations. For example, the sample to be analyzed must be soluble, stable in solution, polar, and relatively clean. These conditions can be arranged in a laboratory, but are difficult to set up in a real, field situation.
MALDI uses a pulsed laser to form ions from a matrix or substrate that is the initial absorber of the photon energy. MALDI is generally used with solid samples, specifically biological samples dissolved and co-crystallized with a UV-absorbing organic compound (matrix) such as nicotinic acid, 3-OH picolinic acid (HPA), 2, dihydroxybenzoic acid (DHBA) or a-cyano-4-hydroxycinnamic acid (CHCA). The mass spectrum of bacteria, virus and spores are generally quite complex, as these are not pure compounds but mixtures.
Because it is not known a priori which compounds from a microorganism will be desorbed, ionized and recorded in the mass spectra, interpretation is not at all straightforward One approach utilizes the different mass spectral patterns observed for different microorganisms in the development of a “library” to which unknown agent can be compared.
The MALDI-MS technique is based on the discovery that desorption/ionization of large, nonvolatile molecules such as proteins and the like can be made when a sample of such molecules is irradiated after being co-deposited with a large molar excess of an energy-absorbing “matrix” material, even though the molecule may not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the molecule of the sample. The ionized molecules are accelerated toward a detector through a flight tube. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass. Thus low-mass ions have a shorter time of flight (TOF) than heavier molecules.
Accordingly, matrix-assisted laser desorption/ionization (MALDI) is most often used with a time-of-flight mass spectrometer, though interestingly, the earlier TOF instruments had pulsed electron impact source. While initially regarded as low mass range, low mass resolution instruments, time-of-flight (TOF) mass spectrometers now provide excellent mass resolution and mass ranges for proteins that extend into the hundreds of kilodaltons.
An exemplary TOF mass spectrometer carrying out MALDI is illustrated in FIG. 2 and operates in the following manner. Samples are deposited as solid solutions in an organic matrix on a sample plate or probe 20. The energy from a short (100 ps to 1 ns) pulsed laser 28 is absorbed by the matrix, resulting in desorption and ionization of sample molecules in the source region. The electric potential between the sample plate 20 and extraction grids 22 results in the acceleration of the ions forming an ion beam 26 into a drift region 24 with kinetic energies of eV+½ mv2, where V is the total accelerating potential, m is the mass of the ion, e the charge, and v the velocity. The flight time of an ion through the drift region 24 having a length D is:t=(m/2 eV)1/2D 
Thus, the flight time is proportional to the square root of the ion's mass/charge ratio. In actuality, the flight time is more complex, reflecting the different times to, initial energies U0, and initial positions s0 when the ions are formed, and is described as follows:T=[(2 m)1/2/eE][(Uo+eEs)1/2±Uo1/2]+{[(2 m)1/2D]/2(Uo+eEs)1/2}+to 
Because t0, U0 and s0 are distributions, the peaks representing a particular mass have a finite time width which limits the mass resolution: R=m/Δm=t/2Δt. To somewhat overcome a relatively low resolution in the TOF MS shown in FIG. 2, the drift region is configured to have a substantial distance, which increases the overall dimensions of the this device.
To improve the mass resolution and to reduce dimensions of the TOF MS shown in FIG. 2, a number of means have been developed. One of these is a TOF MS having a reflectron defining a reflecting region or ion mirror 30, as shown in FIG. 3. The reflecting region “d” 30 is a series of lenses that describe a retarding/reflecting electrical field that returns the ions along a path back toward the source. The reflecting voltage VR is generally slightly higher than the accelerating voltage V so that ions turn around just short of the back of the reflecting region or reflectron. Ions with the same mass but higher kinetic energies have higher velocities and spend less time in the drift region; however, they penetrate the reflecting region 30 more deeply and spend more time there. Thus, the total time spent by ions of different energies in forward L1 and reverse L2 directions of the drift region, and the reflecting region “d” 30, is given by:t=(m/2eV)1/2[L1+L2+4d]
Accordingly, the total time in the TOF MS provided with the reflectron is more nearly the same for ions of different kinetic energies than for the simpler linear time-of-flight instrument shown in FIG. 2. However, the single-stage reflectron shown in FIG. 3 provides only first order correction for the kinetic energy. Higher order energy corrections are possible using dual-stage, quadratic and other non-linear reflections.
Still a further technique improving mass resolution includes using pulsed extraction. The technique involves a short delay time between ionization and ion extraction that permitted ions to drift in the field-free on source. Upon application of the extraction pulse, the more energetic ions will be closer to the source exit and will move through a shorter portion of the accelerating field.
A further approach described to correct the mass dependence includes the TOF instrument, as discussed in reference to FIGS. 2 and 3 and, in addition, configured to use orthogonal extraction/acceleration of ions, as shown in FIG. 4. In this approach, the laser beam 30 irradiates a sample producing ions, which are tightly focused and accelerated along a direction 32 between a pair of electrodes (lens and collimator) 31, so that the distribution in their velocities (arising from their kinetic energy distribution) lies entirely along this direction. The ions are then directed into a volume from which they can be extracted in a direction 34 orthogonal to their initial direction 32.
To even further improve characteristics of the spectrometer, ions may also be additionally focused using the reflectron, which defines the reflecting region 30. Further means directed to improvement of mass resolution may include an RF quadruple ion guide diagrammatically shown as 29 in FIG. 5 and operative to improve focusing of the initial ion beam when a low pressure (1 mTorr) insert gas is used to promote collisional cooling of the ion velocities. The ion guide also makes it possible to utilize high-pressure (1-100 mTorr) sources, or atmospheric pressure sources via a capillary inlet. Because the quadrupole ion guide effectively cools ion kinetic energies, the ions entering the extraction chamber have no memory of their initial kinetic energies. Thus, the orthogonal acceleration mass spectrometer with an R ion guide may be used with almost any ionization source including ESI, MALDI, atmospheric pressure MALDI and EI. A disadvantage of the RF ion guide is that it has a limited mass/charge range. Thus, it has been most successfully used for the low mass ions produced by EI or with high mass multiply-charged ion species produced by ESI.
Improvement of the mass resolution is not the only problem associated with mass spectrometers. As briefly mentioned above, the geometry of the mass spectrometer is also very important Commercial time of flight mass spectrometers generally have drift lengths of the order of 1 meter or longer. Time-of-flight instruments have been miniaturized, specifically for the analysis of biological agents. Provided that the instrument dimensions can sustain high voltage, there is no loss of mass range or sensitivity, but the mass resolution is generally considerably less. For example, in the miniature instrument shown in FIG. 5, the drift length 40 is 3 inches and is floated at the potential of a dual channel plate detector. The sample plate 42 is pulsed to approximately 10 kV giving ions a total energy at the detector of approximately 11.keV. Mass resolutions of up to one part in 1200 have been obtained on this instrument for purified peptides. Mass resolution is less for the more complex biological mixtures that constitute bacteria, virus, and spores.
Thus, many of the known mass spectrometers utilizing various ionization methods may have, among others, the following limitations:                selective mass resolution ability resulting in efficient detection of only a narrow group of biological and chemical agents; and        substantial geometrical dimensions limiting the practical use of at least some of the mass spectrometers.        
These problems were addressed by utilizing interchangeable ionization sources in commercial mass spectrometers. Commercial magnetic and multiple sector instruments have generally been offered with interchangeable electron impact (EI), chemical ionization (CI), field desorption (FD) and fast atom bombardment (FAB) sources. The quadruple-based gas chromatography/mass spectrometer (GCMS) is generally equipped with EI and CI sources. Combination EI/CI sources have also been available, with the ability to select the ionization mode without physically changing the source. Examples of the above may be found in U.S. Pat. No. 5,668,370 which discloses a plurality of ion EI and CI sources, which operate in a mutually exclusive manner and U.S. Pat. No. 6,326,615 which discloses glow discharge and photo ionization sources functioning only simultaneously.
More recently, interchangeable (alternately operating) MALDI and ESI sources have become available for the time-of-flight, Fourier transform mass spectrometer (FTMS), the ion trap (ITMS) and hybrid instruments using combinations of quadruples or ion traps with a time-of-flight mass analyzer. Some of the known structures are configured to have these sources placed in different locations: the electrospray at the atmospheric side of the vacuum chamber, and the MALDI at an intermediate (milliTorr) region. However, both of these ionization techniques are utilized with non-volatile samples and would therefore not address the needs of combined chemical/biological agent detection. In addition, they have not offered the opportunity, or any perceived advantage, for simultaneous operation.
Furthermore, attempts to incorporate EI and Infrared laser desorption (IRLD) ionization sources in a single instrument were undertaken in the past. As disclosed by Dr. Robert J. Cotter, one of the inventors of the present invention, in “Time Resolved Laser Desorption Mass spectrometry”, In. J. Mass Spectrom. Ion Phys. and Ion Processes, pages 49 and 54, respectively (1983), a combination of EI and IRLD was used to resolve some ionization and fragmentation mechanisms, but not analytically.
Furthermore, the mass range and resolution of the instrument, as disclosed in these publications, was limited for the following reasons. First, as is known, since there is no matrix in IRLD, very large ions remained undetected. Second, the IRLD and EI sources were used alternately. As a result, if used to detect biological agents, each of these sources would exhibit specific limitations, as discussed in detail above.
A need, therefore, exists for a single mass spectrometer addressing the full breadth of chemical and biological agents including volatile/non volatile samples, gas and solid phase samples, and configured to carry out a combination of EI and MALDI ionization methods appropriate to the full spectrum of molecular masses.